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Open AcceResearchA novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticlesYogesh Nangia†1, Nishima Wangoo†1, Nisha Goyal1, G Shekhawat2 and C Raman Suri*1

Address: 1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160036, India and 2Institute of Nanotechnology, Northwestern University, Evanston, IL, USA

Email: Yogesh Nangia - yogesh.nangia@imtech.res.in; Nishima Wangoo - nishima@imtech.res.in; Nisha Goyal - nishagoyal@imtech.res.in; G Shekhawat - g-shekhawat@northwestern.edu; C Raman Suri* - raman@imtech.res.in

* Corresponding author †Equal contributors

AbstractBackground: The synthesis of gold nanoparticles (GNPs) has received considerable attentionwith their potential applications in various life sciences related applications. Recently, there hasbeen tremendous excitement in the study of nanoparticles synthesis by using some naturalbiological system, which has led to the development of various biomimetic approaches for thegrowth of advanced nanomaterials. In the present study, we have demonstrated the synthesis ofgold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines inIndia. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization viacharge capping was investigated.

Results: A bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as anovel strain of Stenotrophomonas malophilia (AuRed02) based on its morphology and an analysis ofits 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of goldnanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size.Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are cappedwith negatively charged phosphate groups from NADP rendering them stable in aqueous medium.

Conclusion: The process of synthesis of well-dispersed nanoparticles using a novel microorganismisolated from the gold enriched soil sample has been reported in this study, leading to thedevelopment of an easy bioprocess for synthesis of GNPs. This is the first study in which anextensive characterization of the indigenous bacterium isolated from the actual gold enriched soilwas conducted. Promising mechanism for the biosynthesis of GNPs by the strain and theirstabilization via charge capping is suggested, which involves an NADPH-dependent reductaseenzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process.

BackgroundSynthesis of GNPs and subsequent linkage to biomole-cules has contributed immensely in various life sciences

related applications such as drug-delivery, gene transfer,bioprobes in cell and tissue analysis for visualization ofmicro- and nano-objects, and for observation of the bio-

Published: 20 July 2009

Microbial Cell Factories 2009, 8:39 doi:10.1186/1475-2859-8-39

Received: 27 March 2009Accepted: 20 July 2009

This article is available from: http://www.microbialcellfactories.com/content/8/1/39

© 2009 Nangia et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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logical processes at nano-scale etc. [1-5]. These nanoparti-cles, in general, are synthesized using a number ofsynthetic procedures in various polar and non-polarmedia [6-8]. Recently, there has been tremendous excite-ment in the study of nanoparticles synthesis by usingsome natural biological system. This has led to the devel-opment of various biomimetic approaches for the growthof advanced nanomaterials. Microorganisms, such as bac-teria, yeast and fungi, are known to produce inorganicmaterials either intra- or extracellularly [9-12]. Thesemicroorganisms play an important role in remediation ofmetals through reduction of metal ions. Some of thesemicroorganisms can survive and grow even at high metalion concentrations. They are often exposed to extremeenvironmental conditions, forcing them to resort to spe-cific defense mechanisms to quell such stresses, includingthe toxicity of foreign metal ions or metals [13]. The tox-icity of metal ions is reduced or eliminated by changingthe redox state of the metal ions and/or precipitation ofthe metals intracellularly, thus, forming the basis of thesynthesis of nanoparticles [14]. However, the actualmechanism for the biosynthesis of GNPs by differentmicroorganisms and their stabilization via charge cappingis still not well understood. It was shown in one of the ear-lier studies that the possible mechanism of biosynthesis ofsilver nanoparticles might involve the reduction of silverions due to the electron shuttle enzymatic metal reductionprocess. The enzyme involved in the synthesis of silvernanoparticles may be nitrate reductase present in microor-ganism, which may be induced by the nitrate ions andreduced silver ions to metallic silver [15]. Duran et al alsosuggested the possible mechanism of biosynthesis of sil-ver nanoparticles by Fusarium oxysporum strains (fungi).They proposed the involvement of enzymatic electronshuttle relationship for the formation of Ag+ ions and thesubsequent formation of silver nanoparticle [16].Although there are reports on the biochemical stepsinvolved in metallic nanoparticles synthesis by microor-ganisms [17-20], there are virtually no reports availablewhich may elucidate enzymatic basis of gold nanoparticlesynthesis by Stenotrophomonas maltophilia.

In this paper, we report the rapid synthesis of GNPs byStenotrophomonas maltophilia (Acc No GQ220749), a novelbacterial strain isolated from soil samples from the Singh-bhum gold mines (located in the Jharkhand state ofIndia). A possible mechanism of biosynthesis of gold nan-oparticles from gold chloride (HAuCl4) involves the roleof specific NADPH-dependent reductase enzyme presentin the organism that converts Au3+ to Au0 through electronshuttle enzymatic metal reduction.

Results and discussionIsolation and characterization of strain capable of synthesisizing gold nanoparticlesPure colonies isolated from the gold enriched soil werecharacterized for their morphological and physiologicalcharacteristics by various biochemical tests using theBergeys Manual of Determinative Bacteriology [21] assummarized in Table 1. The screened strain is aerobic,motile, mucoid and yellow in Nutrient Agar medium(Himedia Labs, India). SEM micrographs revealed that thecells of strain AuRed02 are oval shaped, with discretelipopolysaccharides layers on its surface causing the bac-teria to glue to each other as evident from SEM imaging(Fig. 1). No production of acid was noted from theselected carbohydrates such as adonitol, arabinose, fruc-tose, galactose, inositol, inulin, lactose, mannitol, man-nose, rhamnose, sorbitol and xylose. The isolated strainnamed as AuRed02 produced acid from maltose, dextroseand trehalose, showing typical characteristics of Stenotro-phomonas maltophilia. The strain gave a positive lipolyticactivity and was able to utilize citrate as a carbon source.

The 16S rDNA sequence analysis revealed that strainAuRed02 clustered homologue being Pseudomonas Sp.(Acc. No. FJ211222). At species level, strain AuRed02shows the highest level of sequence similarity with Steno-trophomonas maltophilia strain: BL-15 (99%) (AB194325)as shown in phylogenetic tree (Fig. 2).

Characterization of gold nanoparticles synthesized by Stenotrophomonas maltophiliaA solution of gold chloride in a suspension of cell mass ofStenotrophomonas maltophilia changed progressively fromlight yellow to cherry red at temperature 25°C showingformation of gold nanoparticles. Control experiments,without the addition of biomass as well as with heat-killed cells, showed no change in color of suspension (Fig.3a) confirming the formation of GNPs in the presence ofbiomass only. The kinetics of the reaction was studiedusing UV-vis spectroscopy by recording spectra from thecolloidal gold solutions obtained after disrupting thecells, which were resuspended with HAuCl4 solution fordifferent time intervals. The spectra revealed a strongabsorption at nearly 530 nm after 8 hrs of incubation at25°C, gradually showing a red shift with time at 25°C(Fig 4 curves bottom to top). The intense plasmon reso-nance band indicated the formation of spherical goldnanoparticles of approximately 40 nm in diameter [18].Size distribution of GNPs was confirmed by coating adrop of the supernatant solution of disrupted cell suspen-sion by TEM imaging. The images demonstrate gold nan-oparticles possessing an average diameter of 40 nm (±15%) as depicted in (Fig. 3b). Cryo TEM imaging of thethin sections of stained AuRed02 strain after reacting thebiomass with gold chloride solution for 8 h showed the

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presence of gold nanoparticles on the inner cytoplasmicmembrane (see Additional file 1, S1). It is likely that somegold ions (Au3+) cross the cell barrier through ion-trans-port channel and are reduced by the enzymes present onthe cytoplasmic membrane and within the cytoplasm.Energy dispersive spectroscopy analysis (EDS) confirmedthe presence of gold nanoparticles in the suspension. Thespectra (Fig. 3c) present major Au peaks at approximately2 keV besides several other peaks of C, O, and Si whichmight be due to the chemical composition of the samplesubstrate used in the EDS analysis. These results show thatthe strain Stenotrophomonas maltophilia could effectivelysynthesize GNPs of different sizes by resuspending thebiomass for different time intervals in the presence ofHAuCl4 (Fig. 4). The colored solution of GNPs remainedstable for more than 2-weeks of storage at 4°C indicatingthe capping of GNPs with some charged groups. FTIRanalysis of these GNPs further confirmed the capping ofGNPs by phosphate groups (900 cm-1) (see Additional file1, S2).

Promising mechanism aspects of biosynthesis by Stenotrophomonas maltophiliaIn microorganisms, the possible mechanisms of resistanceagainst metal ions usually involve biosorption, bioaccu-mulation, extra-cellular complexation, efflux system,alteration of solubility and toxicity via reduction or oxida-tion [22]. Our study suggested that the biosynthesis ofGNPs and their stabilization via charge capping in Steno-trophomonas maltophilia involved NADPH-dependentreductase enzyme that converts Au3+ to Au0 through elec-tron shuttle enzymatic metal reduction process. For fur-ther confirmation, biomass was incubated with varyingconcentrations of NADPH (from 0.05 mM to 0.8 mMNADPH) and change in color of solution was monitored

Table 1: Biochemical tests of the isolated strain Stenotrophomonas maltophila AuRed02

Gram staining Negative

Catalase test Positive

Citrate test Positive

Oxidase test Negative

Glucose o/f test Negative

Methyl red test Negative

Gelatin test Positive

Carbohydrate utilization tests

Adonitol Negative

Arabinose Negative

Cellobiose Positive

Dextrose Positive

Fructose Negative

Galactose Negative

Inositol Negative

Inulin Negative

Lactose Negative

Maltose Positive

Mannitol Negative

Mannose Negative

Rhamnose Negative

Salicin Positive

Sorbitol Negative

Sucrose Positive

Trehalose Positive

Xylose Negative

SEM image of Stenotrophomonas maltophilia cellsFigure 1SEM image of Stenotrophomonas maltophilia cells. Scale bar corresponds to 1 μm. The cells were imaged after reaction with gold chloride solution for 8 hrs.

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Phylogenetic tree made in MEGA 3.1 software using Neighbor joining methodFigure 2Phylogenetic tree made in MEGA 3.1 software using Neighbor joining method.

(A) GNPs synthesised by Stenotrophomonas maltophilia (tube A)Figure 3(A) GNPs synthesised by Stenotrophomonas mal-tophilia (tube A). Control experiments: gold chloride incu-bated without the biomass (tube B) and with heat-killed cell mass (tube C). (B) TEM image recorded from a drop-coated film of disrupted cell suspension. Scale bar corresponds to 200 nm. (C) Energy Dispersive Spectra of synthesized GNPs showing Au peaks as major constituent.

UV-vis spectra of GNPs solutions prepared by resuspending the biomass of Stenotrophomonas maltophilia for different time intervals in the presence of 1 mM HAuCl4Figure 4UV-vis spectra of GNPs solutions prepared by resus-pending the biomass of Stenotrophomonas mal-tophilia for different time intervals in the presence of 1 mM HAuCl4.

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spectrophotometrically (Fig 5a) and visually (Fig. 5b).Control experiments, without the addition of either cellfree extract (C1) or cell free extract without NADPH (C2),showed no change in color of suspension. However, addi-tion of NADPH in the cell free extract at varying concen-trations (C3 to C7) showed the synthesis of GNPs withgradual increase in color intensity (Fig. 5b). This confirmsthe formation of GNPs only in the presence of both bio-mass and NADPH. Zeta potential measurements of theGNPs showed a peak at -16.7 mV (see Additional file 1,S3), suggesting capping of GNPs by negatively chargedphosphate ions from NADP. Based on these experimentalfindings, a schematic representation of the potentialmechanism of gold nanoparticles synthesis by Stenotro-phomonas maltophilia through enzymatic reduction is pro-posed (Fig. 6). The enzyme involved in the synthesis ofmetal nanoparticles may be a specific reductase present inmicroorganism, which may be induced by the specificions and reduced metal ions to metallic nanoparticles.

ConclusionThe process of synthesis of well-dispersed nanoparticlesusing a highly efficient microorganism Stenotrophomonasmaltophilia has been reported in this study leading to thedevelopment of an easy bioprocess for synthesis of GNPsof desired size and shape. The results presented demon-strate that a specific NADPH-dependent enzyme presentin the isolated strain reduces Au3+ to Au0 through an elec-tron shuttling mechanism leading to the synthesis ofnearly monodispersed GNPs. This green route of biosyn-thesis of GNPs is a simple, economically viable and aneco-friendly process.

MethodsIsolation and characterization of bacteria from the gold enriched soilSoil samples from the gold enriched sites near famousSinghbhum gold mines, Jharkhand state, India were usedas inoculum, serially diluted and plated onto NutrientAgar media (Himedia, India). The plates were incubatedat 30°C for 24 hrs. The colonies obtained were furthersubcultured on Nutrient Agar supplemented with 1 mMHAuCl4 (Sigma, India) and incubated at 30°C for 24 hrs.In one of the isolates, the light yellow color of HAuCl4changed to wine red indicating that the organism was uti-lizing HAuCl4 for the synthesis of GNPs. The morpholog-ical and physiological characterization of the selectedisolate was carried out by biochemical tests using theBergeys Manual of Determinative Bacteriology [21]. Thecell morphology was investigated using Zeiuss-EVO 40scanning electron microscope (SEM). Further characteri-zation of isolate was done by means of 16S rRNA geneanalyses [23]. The 16S rDNA sequencing was done by M/s Bangalore Genei, India.

To obtain stable gold nanoparticles, the conditions of thecharacterized isolate were optimized for different concen-trations of gold chloride, production media, temperature,pH and time. The characterized isolate was inoculatedinto 50 ml sterile Nutrient Broth (NB) and subsequently1 gm of wet biomass was harvested after 16 hrs of incuba-tion. The biomass obtained was washed thrice with deion-ised water (pH 7.0) and added to a 100 ml Erlenmeyerflask containing 1 mM gold chloride solution prepared inde-ionized water spiked with 400 mg Yeast Extract. TheErlenmeyer flasks were incubated at 25°C for 8 hrs to iso-late the nearly monodisperse spherical gold nanoparti-cles. To isolate the pure GNPs, cells were disrupted using0.2% (v/v) Triton X-100. The disrupted samples were cen-trifuged at 3500 rpm for 15 min at 4°C and washed thricewith deionized water to remove cell-debris. The superna-tant was used for the characterization of GNPs.

(A) UV-vis spectra of GNPs synthesis by adding different concentrations of NADPH in the solution of suspended bio-mass along with HAuCl4 (C3 to C7) and (B) shows the GNPs synthesis by adding different concentrations of NADPH in the solution of suspended biomass along with HAuCl4 (tubes C3 to C7)Figure 5(A) UV-vis spectra of GNPs synthesis by adding dif-ferent concentrations of NADPH in the solution of suspended biomass along with HAuCl4 (C3 to C7) and (B) shows the GNPs synthesis by adding different concentrations of NADPH in the solution of sus-pended biomass along with HAuCl4 (tubes C3 to C7). In controls (C1 and C2), either cell mass (C1) or NADPH (C2) was not added.

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Biosynthesis of gold nanoparticles and their characterizationTo determine the peak time-point of maximum gold nan-oparticles synthesis, the absorption spectra of the super-natants were taken at different time intervals and recordedusing a Hitachi U2800 spectrophotometer. TEM studieswere carried out using Jeol 2100 microscope operating at120 kV accelerating voltage. Samples were prepared byplacing a drop of GNPs solutions on carbon-coated TEMgrids. The films on the TEM grids were allowed to dry for5 min at room temperature before analysis. Energy disper-sive spectroscopy analysis (EDS) was carried out to con-firm the synthesis of gold nanoparticles using FEI E-SEMQuanta 200. Particle size and charge distribution (zetapotential) was analyzed using dynamic light scatteringsystem (Beckman Coulter, USA) by illuminating the col-loidal gold solution with He-Ne Laser (633 nm) in a sam-ple cell.

Competing interestsThe authors declare that they have no competing interests.

Authors' contributionsYN and NG carried out the isolation and characterizationwork of bacterial isolate described in the paper. NW tookpart in the characterization of gold nanoparticles anddescribed the mechanism aspects of biosynthesis. GS didall TEM analysis and CRS coordinated the research as wellas the manuscript preparation. All authors read andapproved the final manuscript.

Additional material

AcknowledgementsThis work was funded by a grant from the joint Indo-Russia Integrated Long Term Programme (ILTP), which we gratefully acknowledge. Authors grate-fully acknowledge Mr Kumar Rajesh for necessary technical support.

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Additional file 1S1: Cryo TEM imaging of the thin sections of stained AuRed02 strain; S2: FTIR analysis of GNPs; S3: Zeta potential measurements of the GNPs.Click here for file[http://www.biomedcentral.com/content/supplementary/1475-2859-8-39-S1.doc]

Proposed synthesis mechanism of GNPs by Stenotrophomonas maltophilia through enzymatic reductionFigure 6Proposed synthesis mechanism of GNPs by Stenotrophomonas maltophilia through enzymatic reduction.

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